Probability of myosin heads binding to the non-regulated actin filaments. (a and b) Successive HS-AFM images of F-actin–HMM complexes, where HMM heads bind between two actin filaments in the presence of 0.2 μM ATP (a, scan area: 150 × 90 nm2, 80 × 48 pixels2) or 2 μM ATP (b, scan area: 150 × 75 nm2, 80 × 40 pixels2). Dashed color boxes indicate upper and/or lower HMM heads bound between two actin filaments. Numbers on each frame show the time in seconds. Related to Videos 1, 2, and 3. Scale bars, 30 nm. (c–e) Probabilities of the individual HMM head binding to the four or five binding sites on the non-regulated actin filaments in the presence of 0.2 μM ATP (c, 1,235 events), 2 μM ATP (d, 567 events) and 10 μM ATP (e, 934 events). The average fractional occupancies by HMM heads for all of the sites for each of the ATP conditions showed a decrease in the occupancy with time (right panels in c–e, n = 4). (f) Frequency distributions for the number of myosin heads attached to four neighboring binding sites along a given actin filament or thin filament over time (n = 8 experiments, ∼35 HMM molecules, 898 events). Mean number (±95% CI) of attached myosin heads relative to the four available binding sites given in the inset text. Full lines and dashed lines in the right panels in c–f represent the regression lines and 95% confidence intervals, suggesting a decline in the number of myosin molecules in time. Related to Videos 1, 2, and 3.
